1. Field of the Invention
This invention relates to a process for the production of mixtures of glyoxylic acid and aminomethylphosphonic acid (AMPA), where glycolic acid and oxygen are reacted in an aqueous solution in the presence of AMPA and catalysts consisting of a genetically-engineered microbial transformant which expresses the enzyme glycolate oxidase from spinach ((S)-2-hydroxy-acid oxidase, EC 1.1.3.15), and catalase (EC 1.11.1.6). The glyoxylic acid/aminomethylphosphonic acid mixtures prepared in this manner are useful intermediates in the production of N-(phosphonomethyl)glycine, a broad-spectrum, post-emergent herbicide.
2. Description of the Related Art
Glycolate oxidase, an enzyme commonly found in leafy green plants and mammalian cells, catalyzes the oxidation of glycolic acid to glyoxylic acid, with the concomitant production of hydrogen peroxide: EQU HOCH.sub.2 CO.sub.2 H+O.sub.2 .fwdarw.OCHCO.sub.2 H+H.sub.2 O.sub.2
N. E. Tolbert et al., J. Biol. Chem., Vol. 181, 905-914 (1949) first reported an enzyme, extracted from tobacco leaves, which catalyzed the oxidation of glycolic acid to formic acid and CO.sub.2 via the intermediate formation of glyoxylic acid. The addition of certain compounds, such as ethylenediamine, limited the further oxidation of the intermediate glyoxylic acid. The oxidations were carried out at a pH of about 8, typically using glycolic acid concentrations of about 3-40 mM (millimolar). The optimum pH for the glycolate oxidation was reported to be 8.9. Oxalic acid (100 mM) was reported to inhibit the catalytic action of the glycolate oxidase. Similarly, K. E. Richardson and N. E. Tolbert, J. Biol. Chem., Vol. 236, 1280-1284 (1961) showed that buffers containing tris(hydroxymethyl)aminomethane (TRIS) inhibited the formation of oxalic acid in the glycolate oxidase catalyzed oxidation of glycolic acid. C. O. Clagett, N. E. Tolbert and R. H. Burris, J. Biol. Chem., Vol. 178, 977-987 (1949) reported that the optimum pH for the glycolate oxidase catalyzed oxidation of glycolic acid with oxygen was about 7.8-8.6, and the optimum temperature was 35.degree.-40.degree. C.
I. Zelitch and S. Ochoa, J. Biol. Chem., Vol. 201, 707-718 (1953), and J. C. Robinson et al., J. Biol. Chem., Vol. 237, 2001-2009 (1962), reported that the formation of formic acid and CO.sub.2 in the spinach glycolate oxidase-catalyzed oxidation of glycolic acid resulted from the nonenzymatic reaction of H.sub.2 O.sub.2 with glyoxylic acid. They observed that addition of catalase, an enzyme that catalyzes the decomposition of H.sub.2 O.sub.2, greatly improved the yields of glyoxylic acid by suppressing the formation of formic acid and CO.sub.2. The addition of FMN (flavin mononucleotide) was also found to greatly increase the stability of the glycolate oxidase.
N. A. Frigerio and H. A. Harbury, J. Biol. Chem., Vol. 231, 135-157 (1958) have reported on the preparation and properties of glycolic acid oxidase isolated from spinach. The purified enzyme was found to be very unstable in solution; this instability was ascribed to the relatively weak binding of flavin mononucleotide (FMN) to the enzyme active site, and to the dissociation of enzymatically active tetramers and/or octamers of the enzyme to enzymatically-inactive monomers and dimers, which irreversibly aggregate and precipitate. The addition of FMN (flavin mononucleotide) to solutions of the enzyme greatly increased its stability, and high protein concentrations or high ionic strength maintained the enzyme as octamers or tetramers.
There are numerous other references to the oxidation of glycolic acid catalyzed by glycolate oxidase. The isolation of the enzyme (and an assay method) are described in the following references: I. Zelitch, Methods of Enzymology, Vol. 1, Academic Press, New York, 1955, p. 528-532 (from spinach and tobacco leaves), M. Nishimura et al., Arch. Biochem. Biophys., Vol. 222, 397-402 (1983) (from pumpkin cotyledons), H. Asker and D. Davies, Biochem. Biophys. Acta, Vol. 761, 103-108 (1983) (from rat liver), and M. J. Emes and K. H. Erismann, Int. J. Biochem., Vol. 16, 1373-1378 (1984) (from Lemna Minor L). The structure of the enzyme has also been reported: E. Cederlund et al., Eur. J. Biochem., Vol. 173, 523-530 (1988), and Y. Lindquist and C. Branden, J. Biol. Chem., Vol. 264, 3624-3628, (1989).